Mouse Epigenetics and Nuclear Signaling ELISA Kits
Mouse NF-kB p65 (Nuclear Factor-kB p65) ELISA Kit (MOES01305)
- SKU:
- MOES01305
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q04207
- Sensitivity:
- 0.19ng/mL
- Range:
- 0.31-20ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- NFKB3, p65, RelA
- Reactivity:
- Mouse
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Epigenetics and Nuclear Signaling
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Mouse NF-kB p65 in samples. No significant cross-reactivity or interference between Mouse NF-kB p65 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse NF-kBp65. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse NF-kBp65 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse NF-kBp65, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse NF-kBp65. The concentration of Mouse NF-kBp65 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | NFkB-p65: a subunit of NF-kappa-B transcription complex, which plays a crucial role in inflammatory and immune responses. The inhibitory effect of I-kappa-B upon NF-kappa-B in the cytoplasm is exerted primarily through the interaction with p65. P65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. There are five NFkB proteins in mammals (RelA/NFkB-p65, RelB, c-Rel, NF-_B1/NFkB-p105, and NF-_B2/NFkB-p100). They form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Three splice-variant isoforms have been identified. |
| UniProt Protein Details: | Protein type:Transcription factor; DNA-binding; Nuclear receptor co-regulator Cellular Component: cytoplasm; cytosol; nucleus; protein complex; transcription factor complex Molecular Function:actinin binding; chromatin binding; chromatin DNA binding; DNA binding; enzyme binding; histone deacetylase binding; identical protein binding; NF-kappaB binding; phosphate binding; protein binding; protein complex binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; protein N-terminus binding; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription activator binding; transcription factor activity; transcription factor binding; ubiquitin protein ligase binding Biological Process: activation of NF-kappaB transcription factor; cytokine and chemokine mediated signaling pathway; defense response; hair follicle development; I-kappaB kinase/NF-kappaB cascade; inflammatory response; innate immune response; liver development; negative regulation of apoptosis; negative regulation of insulin receptor signaling pathway; negative regulation of protein catabolic process; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; organ morphogenesis; positive regulation of cell proliferation; positive regulation of chondrocyte differentiation; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of interleukin-12 biosynthetic process; positive regulation of Schwann cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of inflammatory response; regulation of transcription, DNA-dependent; response to bacterium; response to cytokine stimulus; response to muramyl dipeptide; response to organic substance; response to UV-B; transcription from RNA polymerase II promoter; transcription, DNA-dependent |
| UniProt Code: | Q04207 |
| NCBI GenInfo Identifier: | 417926 |
| NCBI Gene ID: | 19697 |
| NCBI Accession: | Q04207. 1 |
| UniProt Secondary Accession: | Q04207,Q62025, |
| UniProt Related Accession: | Q04207 |
| Molecular Weight: | 59,061 Da |
| NCBI Full Name: | Transcription factor p65 |
| NCBI Synonym Full Names: | v-rel reticuloendotheliosis viral oncogene homolog A (avian) |
| NCBI Official Symbol: | Rela |
| NCBI Official Synonym Symbols: | p65 |
| NCBI Protein Information: | transcription factor p65 |
| UniProt Protein Name: | Transcription factor p65 |
| UniProt Synonym Protein Names: | Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 |
| UniProt Gene Name: | Rela |
| UniProt Entry Name: | TF65_MOUSE |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 20 | 2.274 2.304 | 2.289 | 2.219 |
| 10 | 1.624 1.658 | 1.641 | 1.571 |
| 5 | 0.877 0.869 | 0.873 | 0.803 |
| 2.5 | 0.431 0.459 | 0.445 | 0.375 |
| 1.25 | 0.238 0.22 | 0.229 | 0.159 |
| 0.63 | 0.178 0.16 | 0.169 | 0.099 |
| 0.31 | 0.119 0.121 | 0.12 | 0.05 |
| 0 | 0.063 0.077 | 0.07 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse NF-kB p65 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse NF-kB p65 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 1.05 | 1.94 | 6.71 | 1.15 | 2.11 | 6.27 |
| Standard deviation | 0.05 | 0.10 | 0.30 | 0.06 | 0.12 | 0.22 |
| C V (%) | 4.76 | 5.15 | 4.47 | 5.22 | 5.69 | 3.51 |
Recovery
The recovery of Mouse NF-kB p65 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 92-107 | 98 |
| EDTA plasma (n=5) | 87-101 | 94 |
| Cell culture media (n=5) | 88-99 | 94 |
Linearity
Samples were spiked with high concentrations of Mouse NF-kB p65 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 91-104 | 98-110 | 93-107 |
| Average (%) | 97 | 104 | 101 | |
| 1:4 | Range (%) | 88-102 | 86-98 | 83-97 |
| Average (%) | 93 | 92 | 88 | |
| 1:8 | Range (%) | 88-104 | 85-97 | 86-98 |
| Average (%) | 96 | 90 | 91 | |
| 1:16 | Range (%) | 90-102 | 83-93 | 84-96 |
| Average (%) | 96 | 88 | 89 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
- Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
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